Black barley is caused by phytomelanin synthesized in lemma and/or pericarp and the trait is controlled by one dominant gene Blp1. The gene is mapped on chromosome 1H by molecular markers, but it is yet to be isolated. Specific-locus amplified fragment sequencing (SLAF-seq) is an effective method for large-scale de novo single nucleotide polymorphism (SNP) discovery and genotyping. In the present study, SLAF-seq with bulked segregant analysis (BSA) was employed to obtain sufficient markers to fine mapping Blp1 gene in an F2 population derived from Hatiexi No.1 × Zhe5819. Based on SNP screening criteria, a total of 77,542 polymorphic SNPs met the requirements for association analysis. Combining two association analysis methods, the overlapped region with a size of 32.41 Mb on chromosome 1H was obtained as the candidate region of Blp1 gene. According to SLAF-seq data, markers were developed in the target region and were used for mapping the Blp1 gene. Linkage analysis showed that Blp1 co-segregated with HZSNP34 and HZSNP36, and was delimited by two markers (HZSNP35 and HZSNP39) spanning 8.1 cM in 172 homozygous yellow grain F2 plants of Hatiexi No.1 × Zhe5819. More polymorphic markers were screened in the reduced target region and were used to genotype the population. As a result, Blp1 was delimited within a 1.66 Mb on chromosome 1H by the upstream marker HZSNP63 and the downstream marker HZSNP59. Our results demonstrated the utility of SLAF-seq-BSA approach to identify the candidate region and discover polymorphic markers at the specific targeted genomic region.

Calmodulin (CaM) and calmodulin-like (CML) proteins are Ca²⁺ relays and play diverse and multiple roles in plant growth, development and stress responses. However, CaM/CML gene family has not been identified in barley (Hordeum vulgare). In the present study, 5 HvCaMs and 80 HvCMLs were identified through a genome-wide analysis. All HvCaM proteins possessed 4 EF-hand motifs, whereas HvCMLs contained 1 to 4 EF-hand motifs. HvCaM2, HvCaM3 and HvCaM5 coded the same polypeptide although they differed in nucleotide sequence, which was identical to the polypeptides coded by OsCaM1-1, OsCaM1-2 and OsCaM1-3. HvCaMs/CMLs were unevenly distributed over barley 7 chromosomes, and could be phylogenetically classified into 8 groups. HvCaMs/CMLs differed in gene structure, cis-acting elements and tissue expression patterns. Segmental and tandem duplication were observed among HvCaMs/CMLs during evolution. HvCML16, HvCML18, HvCML50 and HvCML78 were dispensable genes and the others were core genes in barley pan-genome. In addition, 14 HvCaM/CML genes were selected to examine their responses to salt, osmotic and low potassium stresses by qRT-PCR, and their expression were stress-and time-dependent. These results facilitate our understanding and further functional identification of HvCaMs/CMLs.

Barley semi-dwarf genes have been extensively explored and widely used in barley breeding programs. The semi-dwarf gene ari-e from Golden Promise is an important gene associated with some agronomic traits and salt tolerance. While ari-e has been mapped on barley chromosome 5H using traditional markers and next-generation sequencing technologies, it has not yet been finely located on this chromosome. We integrated two methods to develop molecular markers for fine-mapping the semi-dwarf gene ari-e: (1) specific-length amplified fragment sequencing (SLAF-seq) with bulked segregant analysis (BSA) to develop SNP markers, and (2) the whole-genome shotgun sequence to develop InDels. Both SNP and InDel markers were developed in the target region and used for fine-mapping the ari-e gene. Linkage analysis showed that ari-e co-segregated with marker InDel-17 and was delimited by two markers (InDel-16 and DGSNP21) spanning 6.8 cM in the doubled haploid (DH) Dash × VB9104 population. The genetic position of ari-e was further confirmed in the Hindmarsh × W1 DH population which was located between InDel-7 and InDel-17. As a result, the overlapping region of the two mapping populations flanked by InDel-16 and InDel-17 was defined as the candidate region spanning 0.58 Mb on the POPSEQ physical map. The current study demonstrated the SLAF-seq for SNP discovery and whole-genome shotgun sequencing for InDel development as an efficient approach to map complex genomic region for isolation of functional gene. The ari-e gene was fine mapped from 10 Mb to 0.58 Mb interval.

SWEET (Sugars Will Eventually be Exported Transporter) proteins, an essential class of sugar transporters, are involved in vital biological processes of plant growth and development. To date, systematical analysis of SWEET family in barley (Hordeum vulgare) has not been reported. In this study, we genome-wide identified 23 HvSWEET genes in barley, which were further clustered into four clades by phylogenetic tree. The members belonging to the same clade showed relatively similar gene structures and conserved protein motifs. Synteny analysis confirmed the tandem and segmental duplications among HvSWEET genes during evolution. Expression profile analysis demonstrated that the patterns of HvSWEET genes varied and the gene neofunctionalization occurred after duplications. Yeast complementary assay and subcellular localization in tobacco leaves suggested that HvSWEET1a and HvSWEET4, highly expressed in seed aleurone and scutellum during germination, respectively, functioned as plasma membrane hexose sugar transporters. Furthermore, genetic variation detection indicated that HvSWEET1a was under artificial selection pressure during barley domestication and improvement. The obtained results facilitate our comprehensive understanding and further functional investigations of barley HvSWEET gene family, and also provide a potential candidate gene for de novo domestication breeding of barley.

Sorghum has a strong tolerance to multi-abiotic stresses such as drought, heat, poor soil fertility, and salinity, yet its growth and development are significantly affected by extreme abiotic stresses, ultimately reducing grain yield. Peroxidase (POD) proteins, which are members of the oxidoreductase enzyme family, play an important role in protecting plants against multi-abiotic stress. However, this gene family has rarely been studied in sorghum. In this study, 114 POD gene members (SbPODs) were identified in sorghum and distributed unevenly throughout 10 chromosomes. Phylogenetic analysis clustered these genes into 12 subgroups. Gene structure and motif analyses showed the structural and functional diversity among the subgroups. These genes have undergone 16 segmental duplication and 32 tandem duplication events, indicating that both segmental and tandem duplications are major contributors to the expansion of the SbPOD gene family. Most duplicated SbPODs have undergone purifying selection with limited functional divergence during duplication events. Compared with maize, rice, and Arabidopsis, SbPOD gene family not only occurred the duplication on the whole-genome level, but also existed independent duplication events. The qRT-PCR results showed twenty SbPOD genes exhibited differential expression patterns in leaves under NaCl, H₂O₂ and PEG stress treatments. Of twenty SbPOD genes, most SbPOD genes showed consistently significant downregulation under NaCl, H₂O₂, and 30% PEG6000 stress treatments, except for SbPOD81 and SbPOD26. Under NaCl stress, the expression level of SbPOD81 showed a down-up regulation trend. Under H₂O₂ stress, the expression level of SbPOD81 showed a down regulation trend. Under 30% PEG6000 stress, SbPOD81 maintained down-up regulation trend but quite different under NaCl stress. In contrast, the expression level of SbPOD26 showed consistent down-up regulation trend under both NaCl and H₂O₂ stress, while after 3 h of adaptation under 30% PEG6000 stress, the expression level of SbPOD26 continued to increase. The analysis of cis-elements and conserved domain showed that SbPOD81 contains one W-box, four MYB, and three MYC cis-elements, while SbPOD26 has two W-boxes, four MBS, four MYB, and eight MYC cis-elements. Additionally, SbPOD26 possesses one PAT1 domain, which is absent in SbPOD81 and other SbPOD genes. This structural distinction not only would explain the observed expression divergence but suggests SbPOD26 may perform specialized biological functions distinct from other SbPOD genes. Totally, 114 POD gene members were identified in sorghum by a series of bioinformatics analysis. The expression patterns of some members of SbPOD gene family exhibited significantly difference under various stress conditions. These findings not only provide valuable insights into the evolutionary dynamics of the SbPOD gene family, but also lay a foundation for further studies on the functions of POD genes.

Plant AT-rich sequence and zinc-binding proteins (PLATZs) are a novel category of plant-specific transcription factors involved in growth, development, and abiotic stress responses. However, the PLATZ gene family has not been identified in barley. In this study, a total of 11 HvPLATZs were identified in barley, and they were unevenly distributed on five of the seven chromosomes. The phylogenetic tree, incorporating PLATZs from Arabidopsis, rice, maize, wheat, and barley, could be classified into six clusters, in which HvPLATZs are absent in Cluster VI. HvPLATZs exhibited conserved motif arrangements with a characteristic PLATZ domain. Two segmental duplication events were observed among HvPLATZs. All HvPLATZs were core genes present in 20 genotypes of the barley pan-genome. The HvPLATZ5 coding sequences were conserved among 20 barley genotypes, whereas HvPLATZ4/9/10 exhibited synonymous single nucleotide polymorphisms (SNPs); the remaining ones showed nonsynonymous variations. The expression of HvPLATZ2/3/8 was ubiquitous in various tissues, whereas HvPLATZ7 appeared transcriptionally silent; the remaining genes displayed tissue-specific expression. The expression of HvPLATZs was modulated by salt stress, potassium deficiency, and osmotic stress, with response patterns being time-, tissue-, and stress type-dependent. The heterologous expression of HvPLATZ3/5/6/8/9/10/11 in yeast enhanced tolerance to salt and osmotic stress, whereas the expression of HvPLATZ2 compromised tolerance. These results advance our comprehension and facilitate further functional characterization of HvPLATZs.